Inflammation is a normal response of the body to protect tissues from infection, injury or disease. The inflammatory response begins with the production and release of chemical agents by cells in the infected, injured or diseased tissue. These agents cause inflammation of the tissue, which generate additional signals that recruit leukocytes. Leukocytes destroy infective or injurious agents, and remove cellular debris from damaged tissue. Although inflammation is a normal response of the body to heal itself, if not controlled, it can result in undesirable chronic inflammation.
The vasculature regulates processes that control inflammation. At sites of injury or infection, cells of the vasculature (including smooth muscle cells and endothelial cells (ECs)) release chemical and protein mediators that attract leukocytes to the injured or damaged area. The leukocytes play a key role by migrating from the circulatory system into the infected area to help resolve the acute inflammation. Cell adhesion molecules expressed on the surface of endothelial cells are one of the primary regulators of this process and assist in recruiting the leukocytes to the affected area. Damaged or injured endothelium stimulates the expression of adhesion molecules, which are responsible for the interaction of leukocytes with the endothelial cell.
Hemodynamics and Vascular Inflammation
Originally viewed simply as a passive barrier or insulation, the endothelial lining is now considered to be a multi-functional organ whose health is essential to normal vascular physiology, and whose dysfunction can be a critical factor in the pathogenesis of vascular disease including inflammatory diseases. It serves as both an endocrine and paracrine organ with numerous regulatory functions. Because endothelial cells lie at the interface between the circulating blood and the vessel wall, they reside in a dynamic physical force environment, experiencing both a normal pressure force and a tangential shearing (frictional) force resulting from the flow of blood over the luminal surface. In recent years there has been considerable focus on the influence of this fluid shear stress on vascular endothelial cell biology (Nerem & Girard (1990) Toxicologic Pathology, 18(4)572-582). One of the first demonstrations that the endothelial cell responds to its hemodynamic environment was the observed alignment of endothelial cells in vivo with the direction of blood flow. (Flaherty, et al. (1972) Circ. Res. 30:23). The dramatic impact of shear stress on vascular inflammation is exemplified in the early events in the pathogenesis of atherosclerosis. The nonrandom distribution of early atherosclerotic lesions observed both in the natural disease process in humans and in experimental animal models suggests that hemodynamic influences can contribute to the early pathogenesis (Asakura & Karino (1990) Circ Res, 66:1045-1066; and Ku, et al. (1985) Arteriosclerosis, 5: 293-302). These early lesions are localized to branched and curved segments of large arteries that are characterized by non-directional and relatively low fluid shear stress. (Montenegro & Eggen (1968) Lab Invest. 18(5):586-593). Conversely, the lesion-protected areas of the vasculature are characterized by unidirectional and relatively high fluid shear stress. These observations led to the development of in vitro model systems that mimic the hemodynamic forces experienced by the endothelium in vivo, so that the effects of this important physical force can be studied in depth.
Substantial evidence now exists to support the role of endothelial cells as powerful transducers of its hemodynamic environment, and that such mechanical forces can directly affect endothelial cell biology (Nerem & Girard (1990) Toxicologic Pathology 18(4):572-582; and Nitzan & Gimrone (1995) FASEB J. 9:874-882). One of the most well-studied effects of fluid shear stress on endothelial cell biology is alteration of EC/leukocyte interactions. The altered adhesivity of leukocytes for the EC is due, at least in part, to the regulation of the adhesion proteins intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). The expression of both ICAM-1 and VCAM-1 are regulated by shear stress (Tsuboi, et al., (1995) Biochem. Biophys. Res. Commun. 206(3):988-996; and Medford, et al., (1994) Circulation. 909(suppl I):I-83. Abstract). In addition, other genes (that function in maintaining vasoactivity, redox state, hemostasis, and growth control) whose expression is regulated by shear stress have been described (Chien, et al. ((1998) Hypertension 31:162-169)).
Oxidative Stress and Hemodynamics
It is now well accepted that oxidative stress and oxidant signaling pathways play important roles in vascular dysfunction associated with atherosclerosis and other inflammatory diseases. Indeed, oxidant stress causes changes in inflammatory gene expression (Kunsch & Medford (1999) Circ. Res. 85:753-766). For example, pro-oxidants stimulate the expression of VCAM-1 and MCP-1 on endothelial cells, whereas, antioxidants inhibit oxidant-stress induced expression of these genes. Cells normally contain endogenous proteins that help to reduce the levels of reactive oxygen species and prevent the damaging effects oxidative stress, thus protecting the cell.
It has been suggested that steady, laminar shear stress induces this protective phenotype of the cell, in part, by activating the expression of genes that function to protect the cell from oxidative stress. Several reports have demonstrated that exposure of endothelial cells to shear stress induces the expression of genes that regulate endothelial cell redox homeostasis including Manganese Superoxide Dismutase (MnSOD), catalase, nitric oxide synthase, Heme oxygenase-1 (HO-1) (Topper, et al., (1996) Proc. Natl. Acad. Sci USA. 93:10417-10422; and Inoue N, Ramasam, et al., (1996) Biochem Biophys Res Commun. 79:32-37). One recent study demonstrated that cells exposed to laminar shear stress (LSS) have lower levels of the free radical, superoxide, than cells exposed to oscillatory shear stress (OSS) (De Keulenaer, et al. (1998) Circ Res. 82:1094). DeKeulenaer et al. suggest that steady LSS can induce compensatory antioxidant defense mechanisms by the observed increased expression of SOD and antioxidant defense enzymes whose level of expression adapts to changes in oxidative stress. Therefore, the local hemodynamic environment of the vasculature can help to regulate intracellular oxidant stress by modulating the expression of genes that function in redox homeostasis. In addition, using a shear stress paradigm to identify genes related to cardiovascular disease, six genes were found to be upregulated (U.S. Pat. No. 6,018,025 to Falb). Although these initial studies have identified a few genes regulated by fluid shear stress, the mechanisms that allow endothelial cells to discriminate between the various types of fluid shear stress and thereby express an anti-inflammatory, antioxidative phenotype remains to be elucidated.
The specific nucleotide sequences that comprise the regulatory regions of genes that respond to hemodynamic influences of the cell, and therefore can mediate antioxidant and anti-inflammatory pathways, provide novel targets for modulating their expression. Particularly, PCT Publication No. WO 00/39275 filed by Florence Medical Ltd., et al., discloses a set of vectors comprising multiple shear stress response elements (SSRE) that include HO-1. The SSRE is defined generally as nucleic acids from the regulatory elements of genes that are regulated in endothelial cells through shear stress forces. The SSRE is further defined as an element necessary and/or sufficient to induce (or suppress) gene expression in endothelial cells exposed to shear stress. The invention provides a method for testing compounds for the ability to regulate endothelial cell gene expression, angiogenesis and/or vasculogenesis to treat disorders related to angiogenesis and or vasculogenesis. However, the specific nucleotide sequences that comprise the regulatory elements of genes that respond to shear stress stimuli have not yet been identified.
The Antioxidant Response Element (ARE)
The antioxidant response element (ARE) with underlined consensus sequence TGACNNNGC (SEQ ID NO 23) is now known to be a transcriptional regulatory element. It is found in the 5′ flanking regions of several genes encoding enzymes involved in the phase II metabolism of xenobiotics, including GST, NQO1, and glucuronosyltransferase. (Rushmore, et al., (1991) J. Biol. Chem. 266:11632; and Jaiswal AK (1994) Biochem. Pharmacol. 48:439). The induction of these enzymes by transcriptional activation through the ARE results from cellular exposure to a variety of chemical entities including electrophilic compounds, antioxidants, Michael reaction acceptors, redox-cycling polyaromatic hydrocarbons, quinones and other agents capable of generating free radical species that alter the cellular redox state. The ability of these enzymes to conjugate redox-cycling chemicals is an important protective mechanism against electrophile and oxidative toxicity. Thus, oxidative stress appears to be the principle signal that acts, either directly or indirectly, through a signaling pathway leading to the transcriptional activation of genes encoding these enzymes. Although this antioxidant defense mechanism has been studied extensively as a hepatic detoxification mechanism, it has also been suggested that the ARE pathway can contribute to antioxidant defenses in the lung via activation of heme oxygenase. (Camhi., et al., (1995) Am. J. Respir. Cell Mol. Biol. 13:387).
U.S. Pat. No. 6,120,994 to Tam discloses an ARE, which constitutes the DNA consensus sequence: 5′-RGR AC NNN GCT-3′ (SEQ ID NO 24) (wherein R is A or G). This patent discloses a method of screening for a compound that increases transcription of a mRNA regulated by the ARE. The ARE is present in a DNA construct containing the ARE operably linked to a protein coding sequence. This construct is used in an assay for cellular extracts of transcription products (either mRNA or protein) in the presence and absence of the compound.
The ARE disclosed in the '994 patent is identified as part of the proapoAI gene. This gene encodes apolipoprotein AI, the major protein component of high-density lipoprotein, which is believed to reduce atherosclerotic risk. Thus, increasing the levels of apolipoprotein via induction of ARE-mediated transcription is reported to have beneficial effects in preventing or treating animal or human atherosclerosis and cardiovascular disease. The '994 patent also teaches a system for screening and identifying compounds that increase transcription of an MRNA regulated by an ARE, for example apolipoprotein AI. In addition, the '994 patent provides a method of treating a human being or an animal with such a compound.
The Maf-recognition Element (MARE)
Recently, Kataoka et al. ((2001), J. Biol. Chem. 276 (36):34074) identified a maf recognition element, referred to as “MARE” having the DNA consensus sequence 5′-TGCTGACTCAGCA-3′ (SEQ ID NO 25). Specifically, Kataoka et al. describes the coordinate induction of the genes for gamma glutamyl cysteine synthase (γ-GCS), NQO1 and HO-1 via the activation of MARE by gold(I) compounds (gold(I) drugs). They also identify that the MARE transcription factors, Nrf-2/Small Maf, are activated by Gold(I) drugs.
Gold(I) drugs are in clinical use for treatment of rheumatoid arthritis. Kataoka et al. describes for the first time the molecular mechanism of action of these drugs. The gene products, γ-GCS, NQO1 and HO-1, which were found to be upregulated by gold(I) drugs are known to be involved in the anti-oxidative stress response as well as anti-inflammatory pathways. Thus, they speculate that the gold(I) drugs can protect against inflammation.
Heme Oxygenase (HO-1)
Heme oxygenase is the enzyme that oxidatively degrades protoheme IX to biliverdin and carbon monoxide. In mammals, biliverdin is further converted to bilirubin, an endogenous radical scavenger, through action of biliverdin reductase. (Stocker, et al., (1987) Proc. Natl. Acad. Sci. U.S.A. 84:5918-5922). Three isoforms of heme oxygenase have been identified; however, only HO-1 is inducible. Initial interest in HO-1 focused on its role in heme catabolism, however, recent studies show that HO-1 is highly responsive to oxidative stress and has potent antioxidant properties (Choi & Alam (1996) Am J. Respir. Cell Mol Biol. 15:9-19). When tissues are pre-exposed to HO-1 inducers their damage and/or acute inflammatory responses are markedly attenuated in a variety of models such as carrageenan-induced pleuritits. (Willis, et al. (1996) Nat Med. 2:87-89), oxidant-induced lung injury (Choi & Alam (1996) Am J. Respir. Cell Mol Biol. 15:9-19; and Lee, et al. (1996) Am. J. Respir. Cell Mol. Biol. 4:556), and endotoxin shock (Yet, et al. (1997) J Biol Chem. 272:4296-4301). In addition, HO-1-deficient humans' exhibit enhanced endothelial cell injury in the presence of oxidative stress (Poss & Tonegawa (1997) Proc. Natl. Acad. Sci. USA. 94:10925; and (Yachie, et al. (1999) J. Clin. Invest. 103:129). Recently, Hayashi and colleagues demonstrated that HO-1 attenuates leukocyte-endothelial cell adhesion in vivo through the action of bilirubin (Hayashi, et al. (1999) Circ Res 85:663-671). This can be one mechanism by which HO-1 is protective in inflammation. HO-1 is upregulated through its ARE site by acute complement-dependant inflammatory responses (Willis et al. (1996) Nature Medicine 2:87-90), as well as electrophilic compounds such as phorbol esters and heavy metals (Prestera et al. (1995) Molecular Medicine 1: 827-837).
NAD(P)H:Quinone Oxidoreductase (NQO1& NQO2)
NQO1 is a cytosolic flavoprotein found ubiquitously in eukaryotes. In addition to NQO1, at least one other homolog (NQO2) has been cloned from humans (˜50% identity at the amino acid level). The expression of NQO1 is induced by exposure to many substances including: aromatic compounds, phenolic antioxidants, peroxides, mercaptans, phorbol esters, ionizing radiation, UV light, and hypoxia. The expression of NQO1 is high in liver and also many extrahepatic tissues including kidney, skeletal muscle, lung, heart, and placenta. NQO1 is upregulated through its MARE/ARE site by rheumatic gold compounds Kataoka et al. ((2001), J. Biol. Chem. 276 (36):34074), as well as xenobiotics and antioxidants (Jaiswal (2000) Free Radical Biology and Medicine 29: 254-262).
It is believed that the primary function of NQO1 is to catalyze the obligatory two electron reductive metabolism and detoxification of quinones and their derivatives. Quinones are highly abundant in nature and human exposure to them is extensive. Quinones are found in all burnt organic materials, including automobile exhaust, cigarette smoke, and urban air pollutants. The obligatory two-electron reduction of quinones catalyzed by NQO1 competes with the one-electron reduction of quinones by other enzymes. These single electron reductions of quinones generate unstable semiquinones that undergo redox cycling in the presence of molecular oxygen leading to the formation of reactive oxygen species causing lipid peroxidation, membrane and DNA damage, oxidative stress, cytotoxicity and mutagenicity. NQO1 must compete with the single-electron reducing enzymes and allow for detoxification. NQO1 generally serves to protect the cell from the redox damaging effects of potentially toxic quinones and semiquinones.
Another major function of NQO1 is to maintain coenzyme Q (Ubiquinone) in the reduced form (Ubiquinol) in membranes. Coenzyme Q10 is a lipid-soluble constituent of (cardiac) mitochondrial membranes. CoQ10 functions as an electron carrier in the respiratory chain through its redox-active quinoid moiety. Other properties of Coenzyme Q10 include “membrane-stabilizing” activity and inhibition of membrane lipid peroxidation. Ubiquinols can react with oxygen radicals and thus prevent direct damage to biomolecules and initiation of lipid peroxidation. CoQ10 protects cardiac mitochondria against oxidative stress and reduces the damaging effects of an oxidative insult in isolated hearts. Several lab studies have reproducibly shown that CoQ10 administration facilitates post-ischemic functional recovery and improves myocardial integrity and metabolic status.
Glutathione S-Transferase (GST) and Gamma Glutamyl Cysteine Synthetase (γ-GCS)
Glutathiones play critical roles in intra- and extra-cellular defenses against oxidative damage, electrophiles and inflammatory responses. Glutathione S-transferase, GST, is a Phase II enzyme that catalyzes the S-conjugation of glutathione with reactive species such as electrophilic compounds. γ-GCSs are also involved in glutathione reactions in that γ-GCS is the rate limiting enzyme for glutathione synthesis. Therefore, regulation of γ-GCS is critical to maintain optimal cellular levels of glutathione.
GSTs are upregulated through an ARE site by chemoprotective compounds (Wasserman & Fahl (1997) Proc. Natl. Acad. Sci. U.S.A. 94:5361-5366) as well as antioxidants (Rushmore & Pickett (1990) 265: 14648-53).
Ferritin (Light and Heavy Chain)
The Ferritins are a distinct class of proteins that generally serve to protect the cell against reactive oxidative species. While iron (Fe) is vital to normal cellular function and survival, it must be well contained because it is able participate in the formation of potentially cytotoxic oxidative reactions. Ferritins are Fe chelators thereby providing a safe form of storage for the Fe in the cell.
It is an object of the present invention to provide a composition and method to protect cells from the potentially damaging effects of oxidative stress.
Another object of the present invention is to provide a composition and method to treat acute and/or chronic inflammation.
Yet another object of the present invention is to provide a method to identify compounds and biologic materials that can induce the expression of cytoprotective enzymes and other protective cell products.
It is a further object of the present invention to provide a method to identify compounds that inhibit VCAM-1 expression.